Abstract:
Malaria aminopeptidases are important in the generation and
regulation of free amino acids that are used in protein anabolism
and for maintaining osmotic stability within the infected erythrocyte.
The intraerythrocytic development of malaria parasites is
blocked when the activity of aminopeptidases is specifically inhibited
by reagents such as bestatin. One of the major aminopeptidases
of malaria parasites is a leucyl aminopeptidase of the M17 family.
We reasoned that, when this enzyme was the target of best at in inhibition,
its overexpression in malaria cells would lead to a reduced
sensitivity to the inhibitor. To address this supposition, transgenic
Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase
were generated by transfection with a plasmid that
housed the full-length gene. Transgenic parasites expressed a
65-kDa protein close to the predicted molecule size of 67.831 kDa
for the introduced leucyl aminopeptidase, and immunofluorescence
studies localized the protein to the cytosol, the location of the
native enzyme. The product of the-transgene was shown to be functionally
active with cytosolic extracts of transgenic parasites exhibiting
twice the leucyl aminopeptidase activity compared with wildtype
parasites. In vitro inhibitor sensitivity assays demonstrated that
the transgenic parasites were more resistant to bestatin (EC₅₀ 64µ ᴍ)
compared with the parent parasites (EC₅₀ 25µ ᴍ). Overexpression of
genes in malaria parasites would have general application in the identification
and validation of targets for antimalarial drugs.
